Monoclonal antibodies selectively bind tumor cell differentiating antigens in vitro and in vivo. Since natural effector mechanisms often do not mediate killing of monoclonal antibody bound cells so we have devised methods of linking extremely toxic proteins to the antibodies to selectively kill tumor cells. Two methods of coupling toxic proteins, (e.g., ricin to antibodies), have been used to kill antigen-positive cells in vitro. Ricin has two subunits: the A subunit blocks protein synthesis when in the cytosol, and the B subunit binds galactose groups on all cell surfaces but also facilitates the transport of ricin A chain to the cytosol. 1) Linkage of the ricin A chain to antibodies yields reagents with low nontarget toxicity but target cell toxicity too slow for in vivo applications: 2) and linkage of intact ricin to antibodies results in very potent target cell toxicity but the nontarget cell killing must be prevented by a ligand which blocks ricin B chain to cells. This has limited its application to in vitro situations where 100 mM lactose can block ricin binding. We have succeeded in developing several new approaches to apply immunotoxins in vivo. 1) Cloning toxins, then altering their structure at the gene level to decrease non target cell toxicity; 2) intrathecal administration of immunotoxins for therapy of brain tumors that kill 2-5 logs of tumor cells in animal models; 3) preparation of genetically engineered immunotoxins for clinical trials of human brain tumor patients; 4) prevention of an immune response against immunotoxin with anti-CD4 antibodies; 5) specific deletion of Purkinje cells in rats, guinea pigs, and rhesus monkeys; 6) use of human cytotoxic proteins such as RNase linked to antibodies to selectively target cells; and 7) understanding the mechanism of human RNase neurotoxins.